Method for Monitoring the Immune Response and Predicting Clinical Outcomes in transplant recipients

ABSTRACT

Methods for monitoring the immune response and predicting clinical outcomes for patients on immunosuppressive drugs (such as transplant patients) are provided. The methods are based on the measurement of an intracellular metabolic marker in lymphocytes (such as ATP) as an indicator of a patient&#39;s immune response.

This application claims priority to U.S. Provisional Application60/371,402, the complete contents of which is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to methods for monitoring the immuneresponse in a patient receiving immunosuppressive drugs. In particular,the invention provides methods based on the measurement of metabolicmarkers of activation in immune cells as an indicator of the patient'simmune response. The methods may be used to predict clinical outcomesand determine treatment courses for patients such as transplantrecipients.

2. Background of the Invention

Each year, 55,000 organ transplants are performed worldwide.¹Cumulatively, the number of living organ recipients is now estimated tobe over 300,000. Most of these transplant recipients will remain onimmunosuppressive drugs for the remainder of their lives to preventrejection episodes. Controlled doses of these drugs are required toprevent overmedication, which may leave the patient susceptible toopportunistic infection, increased risk of cancer and cardio-vasculardisease, and drug toxicity effects, or under-dosing, which may lead toshortened graft survival due to rejection episodes.

The two major drugs used in transplant maintenance today are cyclosporin(Novartis) and Tacrolimus¹ (Fujisawa). These drugs are inhibitors ofcalcineurin, a key enzyme involved in T cell activation.² Therapeuticdrug monitoring (TDM) for these two immunosuppressive drugs is routinelyperformed, as prescribed by the manufacturers. However, the amount ofdrug measured in the blood does not directly correlate with the dose ofdrug administered because of individual pharmacokinetic differences.³ Inaddition, the level of drug determined by immunoassay is not correlatedwith immunosuppressive drug efficacy.⁴ Therefore, the main value ofthese TDM tests is the avoidance of toxic levels and monitoring patientcompliance.

There is currently no method available for the direct assessment ofimmune status in transplant recipients.

SUMMARY OF THE INVENTION

The subject invention provides a method of monitoring immune responsesin a patient who is being evaluated as an organ recipient and/or isreceiving at least one immunosuppressive drug. This convenient andreliable method includes the steps of analyzing the immunologicalresponses of a set or subset of lymphocytes from a blood sample bydetermining at least one level of functional activity and comparing itwith the immunological responses of lymphocytes with defined levels ofhuman immunological responses (low, moderate or strong). The immunestatus assessment of the patient is based on this comparison.Immunological responses are ascertained by measuring an intracellularcomponent that is increased if the cells have responded to a stimulus.

In an advantageous embodiment, the patient is a transplant recipient.For example, the patient may be a recipient of a heart, lungs, kidney,pancreas, liver, small bowel or other organs, tissues, or bone marrowtransplant. At least one immunosuppressive drug may be administered, forexample, calcineurin inhibitors, enzyme inhibitors, antimetabolites,lymphocyte depleting drugs, corticosteroids, or other immune modulators.Drug combinations may also be administered. The overall effect of drugson immune responses may be measured using an assay which detectsincreases in an intracellular component, for example ATP, by a mitogenor antigen stimulation and then employing a luciferase assay, or bymeasuring other metabolic intermediates such as NADP, ionic metabolitessuch as Ca²⁺, or proteins involved in cell cycle regulation such asPCNA.

One aspect of the invention is a method for predicting a clinicaloutcome in a patient who is receiving none or one or moreimmunosuppressive drugs. The method utilizes measured ranges oflymphocyte responses derived from a cohort of apparently healthyindividuals as a means of defining normal ranges of reactivities, andincludes the steps of determining at least one value of lymphocyteresponse in a sample of blood from a patient before or afteradministration of immunosuppressive drug(s); determining whether thelymphocyte response of the cells from the patient receiving theimmunosuppressant drug is higher or lower than the range defined forapparently healthy individuals, or falls within it; and providing aguide for therapy and predicting a clinical outcomes based on thecomparison. Clinical outcomes or conditions which may be predictedinclude transplant rejection, over-medication, and infection. Forexample, a lymphocyte response that falls in the low range indicateshigh immunosuppression and may be indicative of over-medication whichmay lead to organ toxicity, infection, or, in the long term, cancer. Alymphocyte response which falls in the strong range indicates a lowimmunosuppressed condition, which may be indicative of infection or acourse leading to organ rejection. Alternatively, a lymphocyte responsewhich falls in the moderate range may indicate that stability of theimmunological response has been achieved and that no changes intherapeutic regimen are warranted at that time.

Another aspect of the invention is to use the assay to monitor patientswho are being weaned from the immunosuppressant drug(s), or formeasuring patient compliance with medication prescriptive instructions.

In yet another aspect, the invention provides a method for assessing thepharmacodynamic impact (physiological effect) of an immunosupressantdrug in a non-transplant patient. The method includes the steps ofdetermining a value of an immune response in at least one sample oflymphocytes from the non-transplant patient; comparing the value withvalues in a reference set comprising ranges of values of immunologicalresponse for lymphocytes; and assessing the pharmacodynamic impact ofthe immunosupressant drug based on a comparison made in said comparingstep. The non-transplant patient may be receiving the immunosupressantdrug for a disease condition such as autoimmunity, inflammation, Crohn'sDisease, lupus erythromatosus, or rheumatoid arthritis. The method willtypically be carried out in order to reduce complications frominfections or cancer in the non-transplant patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematically illustrates an immune cell function assay as setforth in U.S. Pat. No. 5,773,232 which is herein incorporated byreference in entirety. Lymphocytes are stimulated, incubated, and CD4+cells are separated via magnetic separation; cells are washed and lysedto released ATP, which is detected.

FIG. 2: Immune response distributions of apparently healthy adults,transplant recipients and HIV patients. Y axis=ATP (ng/mL). Zones of low(≦225 ng/mL ATP), moderate (>225 ng/mL ATP and <525 ng/mL ATP) andstrong (≧525 ng/mL ATP) immune response are indicated.

FIG. 3: Comparison of functional immune responses at three sites. Yaxis=ATP (ng/mL).

FIG. 4: Comparison of Cylex™ immune cell function response in kidney,liver and pancreas and simultaneous pancreas and kidney (SPK)recipients. Y axis=ATP (ng/mL).

FIG. 5. Immune response versus Tacrolimus therapeutic drug monitoringtrough. X axis Tacrolimus concentration (ng/mL); Y axis=ATP (ng/mL).

FIG. 6. Immune cell response range vs. time since transplant. Y axistracks time since transplant; X axis is % population.

FIG. 7: Case Study 1: Rejection associated with immune cell functiondespite reduced lymphocyte count following induction therapy. Xaxis=time (days); left Y axis, immune response (ATP ng/mL); right Yaxis=number of lymphocytes.

FIG. 8: Case Study 2. Duration of rescue therapy by measurement ofimmune function. X axis=time in weeks; Y axis=immune response (ATPng/mL).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides methods of determining and/or monitoringthe state of an individual's immune system. The methods involvemeasuring the level of metabolic markers of cellular activation in asubset of immune cells as a measure of immune response, and assignmentof the immune response to a standardized range or zone of immunereactivity. The practice of the method thus provides a single time point“snapshot” of the individual's immune response. Alternatively, bymonitoring an individual's immune response at several time points, it ispossible to obtain a complete picture of the immune system's reactivityover time. The methods of the present invention make it possible toobserve, for example, the response of a patient's immune system to amedical procedure and to adjust treatment protocols accordingly. Inaddition, the inventors have discovered that, using the methods of thepresent invention, it is possible to predict certain clinical outcomesrelated to immune system functioning. For example, in a preferredembodiment of the invention, a patient whose immune system is monitoredmay be on an immunosuppressive drug therapy regimen. In someembodiments, the immunosuppressive drug(s) are administered as theresult of an organ transplant. By monitoring the immune response of sucha patient, it is possible to predict a risk of rejection of thetransplanted organ; or to ascertain if the patient is overmedicated, acondition which can contribute to an increased risk of opportunisticinfection, organ toxicity or cardiovascular complication.

Examples of metabolic markers of cellular activation that can bemeasured in the practice of the present invention include but are notlimited to adenosine triphosphate (ATP), nicotinamide adeninedinucleotide phosphate (NADP), proliferating cell nuclear antigen(PCNA), and/or Ca²⁺ ions. Examples of subsets of immune cells in which ametabolic marker can be measured include but are not limited to subsetsof lymphocytes such as CD4, CD3, CD19 and CD56 cells.

In one embodiment of the present invention, the state of the immunesystem is ascertained by measuring the level of ATP present in CD4cells. In order to make an ATP measurement, a sample of whole blood isobtained from the patient. Certain advantages accrue from using wholeblood in the incubation. For example, the immunosuppressive drugs andantigen presenting cells required for cellular activation are retainedin the sample, the cells are maintained in their own plasma, and aninitial prepurification step is eliminated. The whole blood sample isincubated with at least one stimulant. Examples of suitable stimulantsinclude but are not limited to mitogens (for example, the plant mitogensphytohemagglutinin, Concanavalin A or Pokeweed mitogen); alloantigens(which may be, for example, located on a cellsurface, selectivelypurified, or bound to a synthetic surface e.g. plastic); autoantigens(which also may be, for example, located on a cell surface, selectivelypurified, or bound to a synthetic surface e.g. plastic), and foreignantigens such as those which originate from bacteria, viruses, andvarious parasitic organisms. Examples of viruses include but are notlimited to polyoma viruses (e.g. human polyoma virus BK, polyoma virusJC), human papilloma virus or cytomegalovirus (CMV). In preferredembodiments of the invention, the stimulant is a mitogen such as theplant mitogen phytohemagglutin. After sufficient incubation time, theCD4 cells are removed from (i.e. isolated from) the sample. In general,the time for incubation of the whole blood sample and the mitogen is inthe range of hours to days, and preferably in the range of about 0.5-6to about 24-48 hours. The CD4 cells are then removed, e.g. by usingparamagnetic particles containing an antibody specific for CD4 cells.However, those of skill in the art will recognize that removal of theCD4 cells may be carried out by any of a variety of means which are wellknown, such as column separations, panning, or by using plastic orferrous particles with ligands for the immune cell attachment.

The isolated CD4 cells are then lysed, and the level of ATP in thelysate is quantified. Methods of lysis are well known in the art.Detection of ATP can be accomplished by any of several suitable methods,such as enzyme analysis, high pressure liquid chromatography (HPLC), orthin layer chromatography (TLC). However, in a preferred embodiment, theATP is detected by a luciferin-luciferase assay. An alternative is thatthe cells are not lysed, and the activation product is measured insidethe cell via cytometry, colorimetrically or by chemiluminescentreporter.

The inventor's have discovered that the immune response of an individualat any point in time may be classified according to the amount of ametabolic marker of activation in immune cells that is detected. Forexample, a level of ATP that is detected in a patient may be quantifiedversus known standard levels of ATP detected in apparently healthyindividuals and generated using a calibration curve. The invention thusalso provides a system for classifying an immune response as low,moderate, or strong; or alternatively, for assigning an immune responseto a low, moderate, or strong range or zone of reactivity. In oneembodiment of the classification system of the present invention, ATP isthe metabolic marker, and an immune response is low if the level of ATPdetected is 225 ng/mL or less, moderate if the level is greater than 225ng ATP/mL but less than 525 ng ATP/mL, and strong if the level is 525 ngATP/mL or greater. Thus, an individual's immune response may be rankedas low, moderate or strong based on the level of metabolic marker (e.g.ATP) that is detected.

In addition to obtaining a measurement of an individual's immune systemresponse at a single time point, it may frequently be useful to comparemetabolic marker levels obtained at several time points, for example, inorder to monitor the impact of a course of events on an individual'simmune system. For example, ATP levels may be monitored before, duringand after drug therapy, or before and after organ transplant surgery isperformed, in order to monitor changes over time in the immune responseof the patient in response to these medical procedures. This informationregarding the patient's immune status may be useful as an adjunct totherapeutic drug monitoring at any point in the course of therapy inorder to assess the progress of a patient, the suitability of a drugregimen, and to predict clinical outcomes for a patient (see below).

The present invention provides methods of determining and monitoring thestate of a patient's immune system in response to a stimulus. In someembodiments, the patient is one who is receiving or will be receiving animmunomodulating drug or drugs. For example, the patient may be therecipient of an organ such as heart, lungs, kidney, pancreas, liver,bowel, skin, bone marrow or other organs. Further, a transplant patientmay be the recipient of more than one organ, e.g. a “heart-lung”transplant recipient. Alternatively, the transplant may be transplantedtissue. The transplanted tissue or organ(s) may be from any source knownto those of skill in the art, for example, from a live organ donor suchas a relative (e.g. a sibling) or a matched non-related donor; from acadaver; or from a tissue or artificial “organ” that has been developedand/or maintained in a laboratory setting, e.g. tissue or “organs” grownfrom stem cells, or cultured in a laboratory setting from tissue or cellsamples. Alternatively, the patient may be under treatment for anautoimmune disease such as rheumatoid arthritis, lupus, Crohn's disease,psoriasis, etc. Or the patient may be afflicted with an infectiousdisease, such as Human Immune Deficiency Syndrome related viruses(HIV-1), or Hepatitis associated viruses (HCV). Further, the patient maybe a cancer patient. Those of skill in the art will recognize that themethods of the present invention may be used to monitor and/or assessthe immune system of any individual for any reason.

In yet another aspect, the invention provides a method for assessing thepharmacodynamic impact of an immunosupressant drug in a non-transplantpatient. The method includes the steps of determining a value of animmune response in at least one sample of lymphocytes from thenon-transplant patient; comparing the value with values in a referenceset comprising ranges of values of immunological response forlymphocytes; and assessing the pharmacodynamic impact of theimmunosupressant drug based on a comparison made in said comparing step.The non-transplant patient may be receiving the immunosupressant drugfor a disease condition such as autoimmunity, inflammation, Crohn'sDisease, lupus erythromatosus, or rheumatoid arthritis. The method willtypically be carried out in order to reduce complications frominfections or cancer in the non-transplant patient.

Those of skill in the art will recognize that many types ofimmunosuppressive drugs exist that may be administered, the effects ofwhich on the immune system of a patient may be monitored by the methodsof the present invention. Examples include but are not limited toantilymphocyte drugs such as OKT3, Antithymocytegamma globulin (ATGAM),Daclizumab, and Basiliximab (anti IL2R); calcineurin inhibitors such asTacrolimus (Prograf®, FK506) or cyclosporin (Neoral®); antimetabolitessuch as Azathioprine, Cyclophosphamide, and Mycophenolate mofetil;enzyme inhibitors such as Sirolimus (Rapamune), or corticocorticoidssuch as Prednisone, or methylprednisolone (Solumedrol.).

The present invention provides a method of guiding decisions regardingtherapies and of predicting a clinical outcome of a patient receivingone or more immunosuppressive drugs. Possible clinical outcomes include,for example, rejection of the transplanted organ, infection, or organtoxicity. In order to predict clinical outcomes such as these, it isadvantageous to determine an initial level of the immune response asearly in the immunosuppressive drug course as possible in order to startsurveillance of the patient's immune status coincident with or soonafter transplant surgery, but monitoring may begin at any point afterthe administration of the immunomodulating drugs. Subsequent immuneresponses are ascertained and compared to the earlier response, and toeach other. Any given immune response value (ATP ng/mL) can be assignedto a category of a known range of values, and a comparison of changes inmeasured values over time allows the observation of trends in the immuneresponse of the patient. For example, the patient's immune response atany point in time may be classified as low (ATP ng/mL≦225), moderate(ATP ng/mL>225 and <525), or strong (ATP ng/mL≧525), and a trend towarda heightened or diminished immune response can be observed.

In a preferred embodiment, an initial blood sample is obtained andtested prior to organ transplant surgery and before anyimmunosuppressant drug is administered. The immune response value isascertained and compared to the categories of known value ranges (e.g.,low, moderate or strong). Based on these values the initial drug dosemay be maintained within or modified from the usual practice of doseassignment on the basis of patient body weight. For example, atransplant candidate who is determined to be immunosuppressed due to aninfectious disease (e.g. AIDS) may be given a lower or no drug dose,compared to another individual of the same body weight.

In another preferred embodiment, an initial blood sample is obtained andtested prior to organ transplant surgery and before anyimmunosuppressant drug is administered, and another blood sample istested after surgery and after the administration of drugs. By comparingthe values obtained from these samples, medical judgements can be maderelative to the effect of the surgery and drugs on the patientspecifically regarding the immune status. For example, if the valueobtained from the sample obtained subsequent to the first was in a lowerrange than the first, additional testing may be indicated and ormedication doses reduced to avoid the possibility of over medication. Ifthe value obtained from the sample obtained subsequent to the first onewas in a higher range than the first, additional testing may beindicated and or medication doses increased to avoid the possibility oforgan rejection.

In another preferred embodiment, a blood sample obtained and tested atany point after surgery can provide immune status information regardingthe level of immune suppression when the values are compared tocategories of known value ranges. For example, if the value obtained isin the weak range, additional testing maybe indicated and or medicationdoses reduced to avoid the possibility of over medication. If the valueobtained is in the strong range, additional testing may be indicatedand/or medication doses increased, or rescue therapy initiated to avoidthe possibility of organ rejection. Further, if the value is in themoderate range, and particularly if the value does not fluctuatesignificantly (e.g. stays within the same zone) for at least twoconsecutive monthly measurements, this may indicate that stability ofthe immune response has been achieved, and that adjustments to thetreatment regimen are not necessary at that time.

Regarding the frequency at which blood samples are analyzed, those ofskill in the art will recognize that sampling may be done at any pointat which a skilled practitioner (e.g. a physician) deems it to beadvisable. In general, such testing would be carried out at most daily(e.g. during a time when a patient is most at risk) and at least monthly(e.g. during a time when a patient appears to be relatively stable).

In yet another preferred embodiment, multiple blood samples are obtainedand tested at multiple points after the organ transplant surgery andduring the period when immunomodulating drugs are being administered. Anexample of the predictive value of the methods would be the detection,by utilizing the methods of the present invention, of an increase in theimmune response of the patient from the low to moderate to the strongrange over a period of time. The results may be predictive of potentialacute rejection of the transplanted organ, and may warrant, for example:initiation of other confirmatory tests (e.g. organ biopsy or organspecific blood chemistry analyses); an increase in the dose of the drugbeing administered; a rescue therapy with an alternate drug; or a newcombination of drugs. In general, in order to predict potential organrejection, the state of the immune response must be monitored forseveral days, and preferably for about 3-5 days. In the case ofmonitoring ATP, in order to conclude that a risk of organ rejectionexists, the immune response of the patient must show an increase in therange of at least about 50 ng/mL ATP to 100 ng/mL ATP.

On the other hand, an unexpected decrease in the immune response over aperiod of time may be predictive of the risk of developing anopportunistic infection due to over medication. For example, if apatient's immune response declines from the moderate range to the lowrange, this may be indicative of over-medication and warrant theinitiation of further confirmatory tests (e.g. organ biopsy or organfunction analysis, or assays for infectious organisms by PCR), or areduction or change in medication. In general, in order to detectpossible over medication, the state of the immune response must bemonitored for several days, and preferably for about 3-5 days. In orderto conclude that a risk of overmedication exists, the immune response ofthe patient must show a decrease in the range of at least about 50 ng/mLto 100 ng/mL ATP.

The method may further be useful for monitoring a patient's immuneresponse during the standard immunosupressive-therapy phase of “weaning”the patient from the drugs, i.e. the phase during which a patient's drugdosage is lowered as much as possible to reduce the risk of toxicity,while maintaining a low chance of transplant rejection. In particularthis assay is especially valuable for monitoring tolerance protocolswhere the objective is the eventual removal of all immunosuppressivedrugs. For example, drugs doses may be reduced in a patient whose FK506(Tacrolimus) blood drug level is greater than 15 mg/mL, to 6-10 mg ofFK506 per Kg of body weight 2-3 times per week until the desired immuneresponse level is attained. Similarly, the method may also be used toassess patient compliance with prescribed medication regimens.

The method is also of value in monitoring the functional status of theimmune responses of long-term organ recipients, who have been on thesame medication dosages for extended time periods (years). Patients whohave taken immunosuppressive drugs over a long period have been shown tosuffer from over suppression concurrent with extended drug courses.

The methods of the present invention may be used alone as the primarymeans of tracking a patient's progress. More frequently, the methodswill be used in conjunction with and as an adjunct to other means ofassessing a patient's progress, for example, monitoring drug levels inthe blood, organ biopsy, organ specific blood chemistry tests, and thelike.

EXAMPLES Materials and Methods Study Design.

A multicenter study was conducted with a cohort of 155 apparentlyhealthy adults and 127 organ transplant recipients. The inclusioncriteria for healthy adults, living donor candidates, and volunteersconsisted of men and women between the ages of 19 and 64 who wereeligible to donate blood according to established blood donationguidelines. The inclusion criteria for transplant recipients consistedof men and women aged 19 to 64 who were recipients of cadaveric,living-related, or living unrelated kidney, liver, or pancreas organs.Transplant patients were excluded from the trial if they were infectedwith human immunodeficiency virus or if they were more than five (5)years post transplant.

The apparently healthy adult population who were blood donors consistedof 24% (37) females, 68% (105) males and 8% (13) undesignated, with anage range of 20-60 years. The ethnicity of the population was 59% (91)African American, 28% (44) Caucasian, and 13% (20) other. The transplantpopulation consisted of 43% (55) females, and 57% (72) males, with anage range of 20-64 years. The ethnicity of the population was 24% (31)African American, 69% (87) Caucasian, 7% (9) other. The organstransplanted were, 59% (75) kidney, 34% (43) liver, 2% (3) pancreas and5% (6) multiple organs (simultaneous kidney and pancreas).

Patient History.

Transplant patient history included the condition predisposing patientfor transplant, organ(s) transplanted and their source (cadaver orliving related or unrelated), type of immunosuppressive therapy, timerelative to transplant, dosage and blood levels of immunosuppressivedrugs at time of sample collection, gender and age.

Therapy Protocols.

The types of immunosuppressive therapies were not limited during thisstudy. The standard of care protocol at each center for transplantpatients at hospital discharge and during maintenance outpatient visitswas followed. The types of therapy included: induction therapy with OKT3or ATG; calcineurin inhibitors including Tacrolimus (Prograf®) orcyclosporine-A (Neoral®); steroids (prednisolone); and mycophenolatemofetil (MMF) Cellcept®. Use and dosage were based on standard practiceat each center and varied both within and between centers.

Sample Collection.

Two whole blood samples (one each, sodium heparin and EDTAanticoagulated Vacutainer® tubes) (B-D, Franklin Lakes, N.J.) were drawnfrom apparently healthy adults and transplant patients. Samplescollected in sodium heparin vacutainer tubes (green-tops) were used forthe Cylex™ Immune Cell Function Assay ImmuKnow™ and samples collected inEDTA vacutainer tubes (lavender-tops) were used for flow cytometry.Samples were handled and tested according to each manufacturer's packageinsert.

Cylex™ Immune Cell Function Assay⁵ (ImmuKnow™)

Whole blood (100 μl of a 1:4 dilution) was tested in quadruplicate withor without phytohemagglutinin (PHA) (2.5 μg/mL) overnight (15-18 hoursin a CO₂ incubator at 37° C.). Anti-human CD4 monoclonal antibody coatedmagnetic particles (Dynal Biotech A.S.A., Oslo, Norway) were added toimmunoselect CD4 cells from both the stimulated and non-stimulatedwells. After washing the CD4 cells selected on a strong magnet (Cylex™Cat. 1050) a lysing reagent was added to release intracellular ATP. Aluciferin/luciferase mixture was then added to the cell lysate. Within30 minutes after addition of enzyme, the bioluminescent product wasmeasured in a luminometer (PHL Mediators, Austria or Berthold,Maryville, Tenn., or Turner Designs, Sunnyvale, Calif.) (See FIG. 1).The amount of light emitted (emission maximum 562 nm) was compared to astandard curve generated with ATP calibrators (0, 1, 10, 100, 1000ng/mL). The concentration of ATP (ng/mL) in each sample was thencalculated from the calibration curve using an Excel-based programprovided by Cylex™.

Therapeutic Drug Monitoring

The trough levels of cyclosporin or Taerolimus were performed on wholeblood using a microparticle enzyme immunoassay (META) on the IMximmunoassay system according to the manufacturer's instructions (AbbottDiagnostics, North Chicago, Ill.).

Statistical Analysis

The Cylex™ Immune Cell Function Assay ImmuKnow™ results from apparentlyhealthy adults and transplant patients were analyzed by ANOVA ortwo-tailed t Tests⁶ to assess the statistical significance ofdifferences. A double probability plot (modified ROC Analysis) was usedto establish three zones of immune reactivity in the two populations⁷.

Example 1 Cylex™ Immune Cell Function Assay (ImmuKnow™) Principle of theAssay

Successful management of the transplant recipient currently requireslifelong immunosuppression of the patient to avoid graft rejection¹³.While calcineurin inhibitors have dramatically improved graft survival,the patient is at increased risk of drug toxicity, opportunisticinfections and cancer¹⁴. Managing the relatively narrow therapeuticrange of these drugs remains one of the challenges of transplantmedicine. While tests for the trough levels of the major transplantdrugs are routinely performed for patient monitoring, their main valueis the avoidance of toxicity and assessing patient compliance. Prior tothe present invention, no test existed which directly measured thebioactivity of these drugs in the patient at any point in time. Themethods of the present invention were designed specifically to assessthe immune response in patients receiving immunosuppressive drugs.

The immunosuppressive therapy requirements for patients undergoingtransplant are a function of a large number of variables, including theunderlying disease which led to the transplant, degree ofhistocompatibility matching and pre-transplant sensitization, organtype, as well as the individual patients ability to metabolize the drug.Once the transplant is performed and therapy initiated, the trauma ofsurgery, anesthesia, and possibly blood transfusions will collectivelyimpact the patient's net state of immunosuppression.¹⁶ No testsperformed today allow the assessment of the patient's immune statuseither prior to surgery or during the post-transplant period, nowaveraging 10 years. This application describes the results of amulticenter trial which was designed to statistically establish rangesof immune reactivity in recipients of solid organ transplants using anassay for the measurement of T-cell mediated immunity using the Cylex™Immune Cell Function Assay (ImmuKnow™), which is depicted in FIG. 1.

The Cylex™ assay uses a whole blood sample which is stimulated with theplant mitogen, phytohemagglutinin (PHA). Whole blood was deliberatelychosen as the sample to maintain the lymphocytes in the presence of theimmunosuppressive drugs, which are partitioned between the red cells andthe plasma¹⁷. In addition, while the prepurification step is avoided,the cells are also maintained in their own plasma, avoiding theadditional stimulation from foreign serum which would be required toincubate purified cells.

Phytohemagglutinin was the stimulus of choice because transplantpatients may be anergic and not expected to respond to weaker stimuliincluding recall antigens or alloantigens. In addition, sincecyclosporine and Tacrolimus were designed to inhibit total T cellactivity, a broad spectrum mitogen (like PHA) is most appropriate. Amongmitogens, PHA is more potent than Concanavalin A, or pokeweed mitogen.Therefore, in highly suppressed patients, some “breakthrough” responsemight still be expected.

Traditionally, lymphoproliferation (LPA) has been used as an in vitromodel for cell-mediated immunity¹⁸. In healthy adults undergoingvaccination with tetanus, the Cylex™ assay gave comparable results tolymphoproliferation¹⁰. Dose response curves of the PHA response,comparing a whole blood adaptation of LPA, showed greater sensitivity ofthe Cylex™ assay to lower doses of PHA with responses measurable at 24hours vs. 3 days for LPA¹⁰.

LPA has several disadvantages because it requires 3 to 7 days to performand uses radioactive tritiated thymidine¹⁸. Perhaps more importantly,peripheral blood monocytes are purified from the blood prior to culture,thereby removing the red cells in which the major calcineurin inhibitorsare sequestered. Zeevi¹⁷ recently demonstrated that recall responses,alloreactivity, and PHA-induced activation were also suppressed in thewhole blood ATP assay while proliferation in isolated PBMC were stillmeasurable in transplant recipients receiving immunosuppressive therapy.One possible explanation is that removal of red cells and the prolongedincubation time (3-7 days) of LPA allowed the recovery of cells fromimmunosuppression which cannot occur in the human physiological model.Others have since demonstrated (26) that red blood cells from transplantpatients added back to LPA cultures can restore immunosuppressiveactivity in vitro.

Following overnight incubation of blood with PHA, CD4 cells are selectedusing paramagnetic particles (Dynal) coated with a monoclonal antibodyto the CD4 epitope. CD4 positive cells which orchestrate both cellularand humoral immune responses are targeted since the majorimmunosuppressive drugs were designed to specifically inhibit T cellactivation which has been implicated in rejection¹⁹. Both cyclosporinand Tacrolimus inhibit T cell activation by reducing transcription ofIL-2²⁶.

Most immune cell functions depend directly or indirectly on theproduction of ATP⁸. Weir (U.S. Pat. No. 5,773,232 the complete contentsof which is hereby incorporated by reference) originally patented theuse of ATP as a marker of lymphocyte activation⁵. When assessing theimmune status of transplant patients on cyclosporin or Tacrolimus,cellular ATP is an appropriate target because both compounds inhibitmitochondrial respiration, which is a major source of intracellularATP^(20,21).

Therefore, reduced ATP production directly inhibits the cascade of stepsrequired for lymphocyte functionality including transcription ofcytokine mRNA, cytokine production, and ultimately lymphocyteproliferation, which is also largely cytokine dependent. In a directcomparison of the kinetics of ATP production and cytokines, ATP precededthe appearance of most cytokines measured²⁶.

Transplant recipients receiving immunosuppressive therapy are generallyweakly or non-responsive to skin tests and show inhibited cytokineresponses in vitro⁴ (Ahmed et al.). The Cylex™ Immune Cell FunctionAssay (ImmuKnow™) uses the plant lectin phytobemagglutinin (PHA) tostimulate activation of lymphocytes⁵. Since most of the effectorfunctions of immune cells depend upon cellular energy supply⁸, the assaywas designed to measure increases in intracellular ATP followingactivation by mitogenic, recall antigen, or allogeneicstimulation^(9,10,11). Given that the target of the majorimmunosuppressive drugs, cyclosporin and Tacrolimus, is T cell function,CD4 cells were selected for measurement. Both cyclosporin and Tacrolimusare lipophilic drugs which partition in the red cell membranes. Theassay uses a whole blood sample to maintain the presence of the drugduring the incubation. Use of whole blood not only avoids the necessityof pre-purification of lymphocytes but also maintains an environment foreffective antigen presentation. By using whole blood, the patient's ownplasma is present during the overnight incubation, rather than foreignserum (human AB or fetal calf), which might provide exogenousstimulation¹¹. Patient samples may be used up to 30 hours aftercollection. Since incubations are overnight, testing can be batched atthe end of each day.

A schematic representation of the assay is given in FIG. 1. The assaywas used to obtain the results presented in the Examples 2-12.

Example 2 Statistical Characterization of the Immune Response Levels ofHealthy Adults and Immunosuppressed Populations

A population-based study was conducted comparing the immune responsecharacteristics of apparently healthy adult controls and recipients ofsolid organ transplants. As shown in FIG. 2, the apparently healthycontrol population (n=155) gave a stimulation response on average of 432ng/mL ATP. The immune response characteristics of transplant recipients(n=127) were significantly statistically lower (P<0.0001) than healthycontrols by over 150 ng of ATP, and averaged 282 ng/ml ATP. Statisticalcomparison of the two populations using a modified double probabilityplot allowed the description of three zones of reactivity. Ninety-twopercent (92%) of the transplant patients gave immune response ATP valuesof less than 525 ng/mL. Ninety-four percent (94%) of apparently healthypatients gave immune response ATP values of greater than 225 ng/mL. Thisallows the characterization of a patient's immune response at any pointin time as low (≦225), moderate (>225 but <525), or strong (≧525). Alsoshown is the distribution of viral-induced immunosuppressed HIVpatients. This immunosuppressed population was statistically similar totransplant recipients with an average immune response of 287 ng/mL ATP.

Most immunosuppressive drugs are currently administered on the basis ofbody weight¹³. Yet it is clear that the baseline levels of immuneresponse in patients awaiting transplant vary enormously. Today, nomethod is available for assessing the patient's basal immune reactivityor their initial response to therapy. In an effort to better predicttherapeutic efficacy, Kahan²² have proposed that patients undergo atrial dosing regimen prior to surgery. While this may not always bepractical, an assay which measures a global immune response, likePHA-induced activation, can detect the net effect of surgery,anesthesia, transfusions and therapeutic drugs on immunosuppression.Therefore, following transplant, patient's responses were statisticallydistributed at three levels of reactivity by the Cylex™ Immune FunctionAssay: low, moderate or strong. These groupings provide relativemeasures of reactivity, especially when the patient is used as his/herown control for subsequent determinations. Following surgery and theconversion to oral therapy, the status of immune reactivity for eachpatient could be assessed prior to discharge. Once the patient isstabilized in the post-transplant period, the assay has utility as ameasure of compliance with therapy. The Cylex™ ImmuKnow™ assay may alsobe used to measure the immune response during weaning patients of theirimmunosuppressive drugs. Reducing the drug dose results in acorresponding increase in immune response activity.

This Example demonstrates that the distribution of immune responses inhealthy adults as compared to transplant recipients is statisticallydifferent. These differences can be used to categorize a transplantrecipient's immune response as strong, moderate or low in order toassess the relative pharmacodynamic impact of immunosuppressant drugs inthe management of the transplant patient. Similarly, this exampledemonstrates that the technology is further applicable to otherimmunosuppressed populations such as HIV infected individuals.

Example 3 Site-to-Site Comparison

A study was undertaken to ascertain the consistency results obtainedusing the methods of the present invention at several different clinicalcenters. The distributions in FIG. 3 show that there are no significantdifferences in patient immune responses at each of the three clinicalcenters. Site 1 (n=31) had a mean transplant patient immune response of300 ng/mL ATP which was not statistically different from site 2 (n=49)or site 3 (n=47) with mean immune response values of 296 and 256 ng/mLATP respectively.

The mean immune response at two of the three sites falls within themoderate zone. The population of patients at Site 3, which included alarger number of liver transplants, fell slightly below the moderatezone, consistent with the greater net immunosuppression in thesepatients resulting from the aggregate effects of more traumatic surgeryand lower biological functioning.

Despite differences in populations, therapeutic protocols, and type oftransplant, all three sites performed equivalently as determined by theStandard two-tailed t-Test. In addition, each laboratory was equippedwith luminometers, manufactured by different vendors, and the assayswere performed by technologists with a range of prior laboratoryexperience. All three sites demonstrated proficiency in the use of thetest. Traditionally, assays for cell-mediated immunity have beendifficult to standardize. For the Cylex™ Immune Cell Function AssayImmuKnow™, an ATP calibration curve is run on each plate and reagentshave been manufactured according to Good Manufacturing Practices (GMP).

These results demonstrate that the described methodology is applicableto multiple immunosupressant protocols as used by different medicalinstitutions.

Example 4 Gender and Race Variables

The variables of gender and race were also examined. The immuneresponses of male and female transplant recipients as measured by ATPproduction were not statistically different, though males had immuneresponses 53 ng/mL ATP lower than females. This same trend was seen inapparently healthy adults where males gave statistically lower immuneresponses than females (p<0.04). In addition, the immune responses ofAfrican Americans and Caucasians could not be statisticallydistinguished within transplant patient populations or within apparentlyhealthy adult populations respectively. However, on average, AfricanAmericans receive higher doses of immunosuppressive drugs at each of theclinical sites.

In the recent past, the incidence of organ transplant rejection inAfrican Americans has been significantly higher than forCaucasians^(16,23,24,25). This difference has been attributed toincreased metabolism of immunosuppressive drugs by African Americans. Asa result, most protocols for African Americans now reflect the use ofhigher doses of immunosuppressive drugs. All three institutions in thismulti-center trial adhere to such protocols. Therefore, we analyzed thispopulation of transplant patients on the basis of ethnicity. The resultsdemonstrated no statistical difference between African Americans,Caucasians and other ethnic groups among transplant recipients withregard to the average immunosuppression achieved. One of the questionsthat could be asked is whether healthy adults demonstrate differentbaseline responses. Apparently healthy adults also gave equivalentimmune response levels in this assay. Therefore, equivalent functionalsuppression of all ethnic groups has been achieved across the threecenters, as measured by this assay.

Statistical comparisons of immune response levels on the basis of genderwas also made. Among apparently healthy adults, a marginally significantdifference was seen between males and females, with males demonstratingweaker immune responses. Among the transplant recipients, although thedifferences were not statistically significant, males again gave lowerresponses to PFIA in this assay when compared to females.

This Example demonstrates that despite differences in drug dosingprotocols for African Americans and Caucasians, they achieve similarlevels of immunosuppressive impact. By individualizing patientimmunosuppressant drug management, gender and ethnic difference inimmune response can be overcome by changed dosing of medication.Over-medication based on ethnicity alone can be prevented by monitoringthe patient using the Cylex™ assay.

Example 5 Comparison of Cylex™ Immune Cell Function Response Among Typesof Organ Transplanted

When the average immune response is determined for patients receivingkidneys vs. livers or pancreas or simultaneous pancreas and kidney(SPK), kidney recipients exhibit stronger immune responses on averagethan those receiving either liver or pancreas (FIG. 4) (p<0.05). Whilemost protocols prescribe higher doses of immunosuppressive drugs forkidney recipients than liver or pancreas, the liver transplant patientsare generally more seriously ill. An additional explanation for theliver transplant recipients to be more suppressed is that unpairedmetabolism in the liver leads to increased drug levels or prolongedhalf-life and therefore inhibition of immunity.

In this study, kidney transplant recipients gave statistically strongerimmune responses than liver or pancreas (either alone or simultaneouslywith kidney). These results emphasize the importance of measuring thenet immunosuppression of the patient, since it is well documented thattrauma, anesthesia, transfusion and the underlying disease of thepatient, as well as the therapeutic drugs all contribute to theeffective level of immunosuppression. Therefore, the observation thatliver patients, who receive lower doses of immunosuppressive drugs butalso experience more trauma and are generally more sick than kidneypatients, actually demonstrate lower levels of immune reactivity. Theseother factors undoubtedly contribute more in the early post-transplantperiod (3 months) and may account for another observation from thisstudy that greater immunosuppression is measured in the most “stable”post-transplant period, despite the reduction in dosing ofimmunosuppressive drugs.

This Example demonstrates that the ability to measure netimmunosuppression of an allograft recipient by taking into account theeffects of medications, type of allograft, time post-transplant, trauma,and general health is valuable in managing a transplant patientappropriately.

Example 6 Lack of Direct Correlation between Patient Immune ResponseLevel and Therapeutic Drug Level in Blood

Since the majority of patients in this study received Tacrolimustherapy, a comparison was made between the level of Tacrolimus in wholeblood as detected by immunoassay, and the Cylex™ Immune Cell FunctionAssay (see FIG. 5). No correlation (r²=0.02) was observed, emphasizingthe importance of measuring a direct effect of the drug on real timeimmune system parameter.^(4,12)

Perhaps the most important observation from this trial is that the levelof Tacrolimus as measured by immunoassay of whole blood is notcorrelated with the degree of biological immunosuppression as measuredby the PHA-induced ATP levels.

It is well known that because of pharmacokinetics differences betweenindividuals, the does of calcineurin inhibitor administered is notcorrelated with the analytical level measured in the blood. These dataemphasize the importance of having a functional readout ofimmunosuppression. Both Ahmed⁴ and Shulick¹² have provided similar datausing cytokine based measures of T cell function.

This Example demonstrates that the analytical measurement ofimmunosuppressant drug is not an adequate reflection of thepharmacodynamic impact of the drug.

Example 7 Immune Cell Response Range vs. Time Since Transplant

Based on the statistically established cutoffs in this study, theproportion of transplant patients who were low responders (≦225 ng/mLATP) or strong responders (≧525 ng/mL ATP) in this assay, were plottedversus time after transplant (see FIG. 6). In the first two months aftertransplant patients 50% of newly transplanted patients demonstratedimmune cell function ATP levels greater than 525 ng/mL and the smallestproportion (˜30%) gave ATP levels less than 225 ng/mL. Over the next fewmonths, the proportion of patients showing PHA induced ATP levels<225ng/mL increased steadily. In the 6-12 month window, no patients reactedin the strong immune response zone (≧525 ng/mL). The greatest deviationin the low and strong immune responder groups occurred at one year ormore after transplant with a slight upswing in the ATP values ≧525 ng/mLat 4 years.

When the proportion of transplant patients with immune response ATPvalues of ≧525 ng/mL or ≦225/ng/mL are plotted as a function of time, itis not surprising that the greatest number of patients at the extremesof this assay occur in the first months following transplant. In thismulti-center trial, 6-12 months appeared to be the point of greateststability, with no patients with values greater than 525 ng/mL. At laterpoints (>1 year), the proportion of patients with immune response levels≦225 ng/mL increases. It would appear that once patients have recoveredfrom the trauma of surgery, the effectiveness of immunosuppressivetherapy appears to increase over time, despite lower dosing of thesedrugs. This demonstrates again the importance of the measurement of netimmunosuppression, which may indicate that there is additional rationalesupporting individualized titration of immunosuppressive therapy. Theavailability of a direct measure of immune system activity reflectingthe potency of therapy in real time allows for a mechanism for drugadjustment. The assay also readily responds to intervention therapies,so that the relative impact of such therapies can now be calibrated,better reflecting a patient's individualized response to these drugs.Ultimately, tailored therapies offer the promise of reducing thelong-term morbidities associated with the prolonged use of these drugswithout compromising the life of the transplanted organ.

This Example demonstrates that most patients receiving immunosuppressanttherapy for prolonged periods of time (greater than one year) show highlevels of functional suppression of immunity that does not correlatewith the drug level in the blood, which is often at its lowest point.

Example 8 Prediction of Risk of Rejection

Typically, a high dose of an immunosuppressant drug is administered to apatient immediately after transplant surgery, and followed up withadditional doses taken daily. Drug monitoring assays and organ functionassays, (e.g. creatinine) are performed on a monthly basis. The Cylex™Immune Cell Function Assay may be performed concurrently to detectchanges in the immune response over a period of time. For example,results showing a progression in immune response from the low range(≦225 ng/mL ATP), through the moderate range, and into the strong range(≧525 ng/mL ATP) may serve as a cautionary marker of potential acuterejection, permitting the treating physician to increase drug orintroduce a rescue therapy with alternate drug doses, or use incombination with others, or initiate other clinical confirmatory tests(e.g., organ biopsy).

CASE STUDY 1: Induction therapy in transplant recipients leads to adramatic decrease in the total number of circulating lymphocytes and isintended to reduce acute rejection episodes. This Case Study reports apatient's clinical course as monitored by the Cylex™ Immune CellFunction Assay for 1-year following transplantation (FIG. 7).

A 51 year-old Caucasian male with end-stage renal disease (ESRD)secondary to glomerulonephritis received a living relatedhaplotype-matched kidney. The immunosuppression protocol consisted ofinduction with anti-CD52 antibodies and maintenance therapy withSirolimus. The patient's immune response was monitored by the methods ofthe present invention.

To perform the assay, 200 μl of sodium heparin anticoagulated wholeblood was diluted, aliquoted into wells of a 96-well microtiter plateand stimulated overnight with PHA. CD4+ cells were then selected usingantibody-coated magnetic particles, washed and lysed to releaseintracellular ATP. The intensity of the patient's immune response wasquantified by measuring the amount of intracellular ATP produced inresponse to stimulation.

Treatment with anti-CD52 at transplant led to profound lymphocytedepletion, but the intensity of the post-transplant immune responseactually rose from pre-transplant to day 13 see FIG. 7). On day 23, aprotocol biopsy revealed a Banff Ib rejection (in the absence of asignificant rise in creatinine). Rescue therapy consisted of treatmentwith solumedrol, a prednisone taper, and Sirolimus. Following thistreatment, the patient's Immune Response dropped, indicating an increasein the level of immunosuppression. During this period, Sirolimus drugdosing remained constant, creatinine levels were stable, and lymphocytecounts progressively increased.

Thus, initial induction therapy with anti CD52 led to reduction in theabsolute number of circulating lymphocytes. A decrease in the cellnumber, however, did not translate into suppression of the patient'sfunctional immune response. The immune response level after inductionwas increased over the pre-transplant value and was associated withrejection. The patient rejected about a week later, despite dramaticallylower absolute lymphocyte counts, necessitating modifications to thedrug regimen.

The functional responsiveness of circulating lymphocytes is a criticalmeasure in assessing the efficacy of immunosuppressive therapy. Thisexample demonstrates that despite effective lymphodepletion and ongoingimmunosuppressive therapy, the lymphocytes remained metabolically activeas measured by ATP and as reflected in the ensuing rejection event.ImmuKnow™ provides a valuable adjunct to other clinical parameters inthe monitoring of patients post transplant.

CASE STUDY 2. Following rejection, the duration of infusion ofmonoclonal antibody OKT3 (anti-CD3) (7 to 14 days together withsteroids) is based on the degree of rejection as judged by biopsy. Mildto moderate rejections may be given a 7-day treatment while severerejections warrant a 14-day course. This Case Study describes theclinical course of a multivisceral transplant recipient followingtreatment for acute rejection with OKT3 and Solumedrol as a function ofher immune response (FIG. 8).

A 48-year-old African American female was treated with rATG and infusedwith donor bone marrow cells prior to receiving an irradiated,multivisceral transplant (small bowel). She received tacrolimus asmaintenance therapy.

To perform the Cylex™ Immune Cell Function Assay, 200 μl of sodiumheparin anticoagulated whole blood was diluted, aliquoted into wells ofa 96-well microtiter plate and stimulated overnight with PHA. CD4+ cellswere then selected using antibody-coated magnetic particles, washed andlysed to release intracellular ATP. The intensity of the patient'simmune response was quantified by measuring the amount of intracellularATP produced in response to stimulation. Acute rejection was diagnosedby histopathologic studies of random and endoscopically guided multiplemucosal biopsies. To treat the acute rejection, the patient was giveninfusions of OKT3 at 10 mg/day in conjunction with IV tacrolimus andsolumedrol.

Following a period of stabilization after transplant, tapering oftacrolimus was initated. On week 38, the patient was diagnosed with anacute rejection and infused with OKT3/FK and Solumedrol. On week 39, theimmune response as measured by Cylex™ Immune Cell Function Assay wasextremely high (ATP=954 ng/mL, see FIG. 8). An additional three-daycourse of OKT3, instead of 7 days, was given. The immune responsedropped by half (ATP=582 ng/mL) and OKT3 and Solumedrol were stopped.The immune response continued to drop through week 41, but climbed tothe upper end of the moderate zone two weeks later (week 43). Solumedroltreatment was re-initiated and the immune response dropped significantly(week 44). The patient is currently clinically stable.

Once a recipient's immune system begins to reject an allograft,extremely potent immunosuppressants are required to limit damage orpotential loss of the organ. Until recently, the type and dosage ofdrug(s) and the length of time required to rescue an acute rejectionevent were based predominately on biopsy results alone. The Cylex™Immune Cell Function Assay provides a measure of a patient's globalimmune response that incorporates the aggregate impact of multipledrugs, and the patient's clinical condition. As shown in FIG. 8, initialtreatment with OKT3 did not reduce the patient's immune responseadequately. A second treatment was needed, but the usual length of timewas reduced from 7 to 3 days. By supplementing maintenance therapy oftacrolimus with steroids, this patient is now clinically stable with anintact, functional and rejection free organ. The Cylex™ immune functionassay ImmuKnow™ assay gauged the effectiveness of rescue therapy andhelped limit the amount of immunosuppressants administered to thispatient.

The Cylex™ Immune Cell Function Assay provides a measure of theaggregate impact of immunosupressive therapy, allowing the physician tobetter individualize a patient's course of therapy.

Example 9 Prediction of Risk of Infection

A dose of an immunosuppressant drug such as Rapamycin is reduced overtime according to a protocol during post-liver-transplant therapy.Therapeutic drug monitoring assays are performed monthly which indicatethat drug trough levels are within expected range.

The Cylex™ assay performed at the same time detects an unexpectedcontinued decrease in the immune response over an extended period oftime from the moderate range (>225 ng/mL ATP, <525 ng/mL ATP) to the lowrange (≦225 ng/mL ATP). Further monitoring indicates a continuingdecrease over time. These results serve as an indication of potentialrisk of opportunistic infection due to over medication, and allows thephysician to reduce drug doses or initiate other clinical confirmatorytests (e.g., organ biopsy or organ function analysis). See case studies1 and 2.

Example 10 Prediction of Favorable Outcome

An immunosuppressive drug such as Cyclosporin is administered over anextended period of time after heart transplant surgery. During this timethe drug is weaned in order to avoid long-term toxic effects. Drug levelmonitoring reflects this decrease, however the Cylex™ immune functionassay performed concurrently indicates that the patient's immuneresponse remains within the moderate range (>225 ng/mL ATP, <525 ng/mLATP). This indicates that there is little likelihood of rejection ordamage to the organ due to toxicity.

REFERENCES

-   1. UNOS, 2001 International Transplant Directory. Transplant News;    Transplant Communications Inc., Fresno, Calif.-   2. Rovira, P, Mascarell, L and Truffa-Bachi, P. The Impact of Immune    Suppressive Drugs on the Analysis of T Cell Activation. Current    Medicinal Chem. 2000(7):673-692.-   3. Venkataraman, R, Shaw, L M, Sarkozi, L, Mullins, R, Pirsch, J, et    al. Clinical Utility of Monitoring Tacrolimus Blood Concentrations    in Liver Transplant Patients. J. Clin. Pharm. 2001; 41:542-551.-   4. Ahmed, M, Vankataraman, R, Logar, A J, Rao, A S, Bartley, G P,    Robert, K, Dodson, F S, Shapiro, R, Fung J J and Zeevi, A.    Quantitation of Immunosuppression by Tacrolimus Using Flow    Cytometric Analysis of Interleukin-2 and Y-Interferon and Inhibition    in CD8⁻ and CD8+ Peripheral Blood T Cells, Therapeutic Drug    Monitoring 2001; 23(4): 354-362.-   5. Weir, M L Methods for Measurement of Lymphocyte Function. U.S.    Pat. No. 5,773,232: 1998.-   6. Hawkins, D M Diagnostics for Conformity of Paired Quantitative    Measurements, In: Statistics in Medicine: 2001-   7. Rocke, D M and Lorenzato, S. A Two-Component Model for    Measurement Error in Analytical Chemistry, Technometrics, 1995; 37,    176-184.-   8. Buttgereit, F, Burmester, G-R, Brand, M D. Bioenergetics of    Immune Functions. Fundamental and Therapeutic Aspects. Immunol.    Today 2000; 21:192-199.-   9. Britz, J A, Sottong, P. and Kowalski, R. In vitro CMI™: Rapid    Assay for Measuring Cell-Mediated Immunity. In: Luminescence    Biotechnology Instruments and Applications. Ed. Van Dyke, Van Dyke    and Woodfork, CRC Press. 2002; p. 331-344.-   10. Sottong, P R, Rosebrock, J A, Britz, J A and Kramer, T R.    Measurement of T-lymphocyte Responses in Whole Blood Cultures Using    Newly Synthesized DNA and ATP. Clinical and Diagnostic Laboratory    Immunology, 2000; Vol. 7:307-311.-   11. Halsey, J F, New Methods, Clinical Applications of T Cell    Functional Assays. Advance/Laboratory. October 2001; p 67-72.-   12. Schulick, R D, Weir, M B, Miller, M W, Cohen, D J, Bernas, B L    and Shearer, G M. Longitudinal Study of In Vitro CD4+ Helper Cell    Function in Recently Transplanted Renal Allograft Patients    Undergoing Tapering of their Immunosuppressive Drugs,    Transplantation. 1993; Vol. 56 No. 3:590-596.-   13. Danovitch, G M. Current Immunosuppressive Regimes in Organ    Transplantation. Kidney International. 2001. Vol. 59:p 388-402.-   14. Danovitch, G M. Immunosuppressive medications for renal    transplantation: A multiplechoice question. International Society of    Nephrology. Kidney International. 2001 Vol. 59:388-402.-   15. Jusko, W J, Thomson, A W, et al. Consensus Document: Therapeutic    Monitoring of Tacrolimus (FK-506). Ther. Drug Monitoring; 1995;    17:606-614.-   16. Burdick, J F, Shinozuka, N. Immune Monitoring for Transplant    Recipients. In: Kidney Transplant & Rejection: Diagnosis and    Treatment. Ed. Racusen, Dekker, Marcel Inc. 1998; p 563-575.-   17. Zeevi, A, Bentlejewski, C, Spichty, K, Griffith, B, Abu-Elmagd,    K, Hooper, N, Kowalski, R, and Fung, J. Post Transplant Immune    Monitoring—ATP Based Assay for T Cell activation. Presented at 2001    Clinical Histocompatibility Workshop, Honolulu, Hi.-   18. Fletcher, M A, Urban, R G, Asthana, D., Walling, J.,    Friedlander, A., Page, J. B. Lymphocyte Proliferation, In: Rose, N.    R., et al (eds.), Manual of Clinical Laboratory Immunology: 5th Ed.    American Society for Microbiology, 1997; p 313-319. Washington, D.C.-   19. Liu, J, Farmer, JD, Lane, W S, et al. Calcineurin is a common    target of cyclophilincyclosporin A and FKBP-FK506 complexes. 1991.    Cell 66:807-901.-   20. Karlsson H, DePierre J W, Nassberger, L. Energy Levels in    Resting and Mitogen-Stimulated Human Lymphocytes During Treatment    with FK506 or Cyclosporin A In Vitro. 1997. Biochim Biophys Acta.    1319(2-3):301-310.-   21. Horigome, A, Hirano T, Oka, K. Tacrolimus-Induced Apoptosis and    its Prevention by Interleukins in Mitogen-Activated Human    Peripheral-Blood Mononuclear Cells. 1998. Immunopharmacology    39(1):21-30.-   22. Kahan, B D, M. Welsh, L. Rutzky, et al. The Ability of    Pretransplant Test-Dose Pharmacokinetic Proflies to Reduce Early    Adverse Events after Renal Transplantation. Transplantation 1992;    53:345.-   23. Butkus, D E, Meydrech, E F, Raju, S S. Racial Differences in the    Survival of Cadaveric Renal Allografts. Overriding Effects of HLA    Matching and Socioeconomic Factors. N. Engl. J. Med. 1992.    327.-840-845.-   24. Van Buren, D H. Renal Transplantation Outcomes in    African-American Patients. Transplant Immunol. Lett. 1999. 15:61-11.-   25. Nagashima, N, Watanabe, T, Nakamura, M, Shalabi, A, Burdick,    J F. Decreased Effect of Immunosuppression on Immunocompetence in    African-Americans After Kidney and Liver Transplantation, Clin.    Transplantation. 2001. 15:111-115.-   26. Halsey, personal communication.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1-49. (canceled)
 50. A method of assessing an immune response to anantigen stimulant in a patient, comprising the steps of: incubating ablood sample from said patient for 0.5-48 hrs, at least a first portionof said blood sample including an antigen stimulant, and at least asecond portion of said blood sample being non-stimulated; selectivelyseparating a subset of lymphocytes from said first portion and from saidsecond portion of said blood sample, said subset of lymphocytes beingselected from CD3, CD4, CD19, and CD56 cells, said selectivelyseparating step being performed with a substrate having an antibodyspecific for said subset of lymphocytes; lysing said subset oflymphocytes from each of said first portion and said second portion ofsaid blood sample to produce a lysate containing adenosine triphosphate(ATP); quantifying ATP in said lysate for said subset of lymphocytesfrom said first portion and said second portion of said blood sample;and determining if said patient has been previously exposed to saidantigen stimulant based on a difference between said ATP in said lysatefor said subset of lymphocytes from said first portion and said secondportion of said blood sample.
 51. The method of claim 50 wherein saidquantifying step is performed with a luciferin:luciferase assay.
 52. Themethod of claim 50 wherein said substrate used in said selectivelyseparating step is paramagnetic beads.
 53. The method of claim 50wherein said lymphocytes are CD3 lymphocytes.
 54. A method of monitoringan immune response in a patient receiving at least one immunosuppresivedrug, comprising the steps of: at first and second time periods,determining a value of an immune response for said patient whichreflects activation of one of CD3, CD4, CD 19, or CD56 lymphocytes in awhole blood sample stimulated by exposure to a mitogen or antigenstimulant wherein said value is measured in terms of adenosinetriphosphate (ATP) present in cell lysate of said one or CD3, CD4, CD19or CD 56 lymphocytes isolated from said blood sample; comparing saidvalue determined at said first time period with said value determined atsaid second time period, and if there is a decrease of at least about 50ng/ml ATP, determining that said patient is at risk of beingovermedicated with said one or more immunosuppressive drugs.
 55. Themethod of claim 54 wherein said patient has at least one of Crohn'sdisease, cancer, human immunodeficiency virus, inflammation, rheumatoidarthritis, or lupus erythmatosus.
 56. The method of claim 54 whereinsaid patient is an organ or non-organ transplant patient.